This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Metabolic homeostasis, or the ability to closely match energy production with demand, is a fundamental requirement for the health and normal functioning of the cells, tissues, and organs of the body. Therefore, several technologies have been developed to assess cellular energy production. Optical imaging modalities are particularly attractive, because light is minimally invasive, easily delivered to the various tissues of the body, and capable of providing rapid feedback with high spatial resolution. One approach to characterizing the metabolic state has been to use the intrinsic fluorescence emission from the reduced form of nicotinamide adenine dinucleotide (NADH) and flavoproteins that directly participate in mitochondrial energy production. This has been extensively applied to a variety of tissues and has been instrumental in the development of our current understanding of the regulation and maintenance of the metabolic state. However, interpreting the observed changes in tissue fluorescence can be problematic, often requiring assumptions or the need of additional measurements for effective application. Traditionally, metabolic imaging has employed fluorescence intensity as a surrogate for the concentration of electron donors to the respiratory chain. Recent studies, however, have shown that these measurements of fluorophore concentration may be error prone. Fundamentally, this is because the fluorescence intensity is dependent on the local environment of the fluorophore and is properly expressed as a product of both the fluorophore's lifetime and concentration. Since lifetime changes can also occur as a result of changes in the ratio of the free to enzyme-bound fluorophores populations, changes in intensity are difficult to interpret. Other studies have suggested that NADH fluorescence lifetime imaging (FLIM) may provide a more accurate measurement of cellular energetics. While FLIM is a promising, novel alternative, it has yet to be properly evaluated in well controlled, yet realistic cellular environments. Using easily manipulated yet relevant in vitro cultures, we propose to systematically compare measurements of the cellular metabolic state obtained from NADH FLIM with the traditional assessment made using fluorescence intensity alone. By establishing the advantages and limitations of this new technique, we will be able to properly deploy metabolic imaging techniques to better characterize, diagnose and develop treatment interventions for a broad range of human disease.